Role of Cu Transporter Proteins in Atherosclerosis

铜转运蛋白在动脉粥样硬化中的作用

基本信息

批准号：
9590248
负责人：
TOHRU FUKAI
金额：
--
依托单位：
CHARLIE NORWOOD VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-10-01 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9590248
关键词：
3&apos Untranslated Regions ATP phosphohydrolase Address Adhesions Aortic Aneurysm Apolipoprotein E Arterial Fatty Streak Atherosclerosis Binding Biological Assay Biological Availability Biosensor Blood Vessels Bone Marrow Carrier Proteins Cell Adhesion Molecules Cell Nucleus Cells Chelating Agents Chimera organism Copper Data Development Diabetes Mellitus Disease Down-Regulation Electrical Resistance Endothelial Cells Enzymes Exhibits Extracellular Space Fluorescence Microscopy Fractionation Functional disorder Goals Grant High Fat Diet Hypertension ICAM1 gene Image Analysis Immunofluorescence Immunologic Inductively Coupled Plasma Mass Spectrometry Inflammation Inflammatory Inflammatory Response Knockout Mice Label Leukocytes Measures Mediating Micronutrients Migration Assay Molecular Molecular Chaperones Mus NADPH Oxidase Nuclear Nutrient Oxidative Stress Permeability Population Process Protein-Lysine 6-Oxidase Proteins Recruitment Activity Reporter Reporter Genes Reporting Resistance Roentgen Rays Role Small Interfering RNA Synchrotrons Testing Time Tissues Transgenic Organisms Vascular Diseases Vascular Permeabilities Veterans ataxia telangiectasia mutated protein atheroprotective base bioluminescence imaging cadherin 5 chromatin immunoprecipitation copper-transporting ATPase cytokine endothelial dysfunction enzyme activity extracellular in vivo insight intravital microscopy live cell imaging migration mortality mutant new therapeutic target novel prevent promoter public health relevance reconstitution therapeutic target transcription factor treatment strategy vascular inflammation

项目摘要

DESCRIPTION (provided by applicant): Atherosclerosis, which is regulated by inflammation, endothelial cell (EC) barrier dysfunction, and oxidative stress, is the major cause of aortic aneurysm (AA), and mortality in Veteran population. Copper (Cu), an essential nutrient, is involved in normal function while excess Cu contributes to inflammatory diseases including atherosclerosis with unknown mechanism. Since excess Cu is toxic, bioavailability of intracellular Cu is tightly controlled by Cu transporting ATPase (ATP7A) which obtains Cu from cytosolic Cu chaperone Atox1 and then delivers Cu to secretory Cu enzymes including ecSOD, or exports Cu to extracellular space. Our lab reported that vascular ATP7A-ecSOD axis protects against endothelial dysfunction in hypertension and type1 diabetes, and that Atox1 functions as a Cu-dependent transcription factor, when it localizes at nucleus. However, a role of ATP7A and its relationship with nuclear Atox1 in atherosclerosis is entirely unknown. Preliminary data suggest that ATP7A mutant (ATP7Amut) mice which have reduced Cu export function crossed with ApoE-/- mice accelerate atherosclerotic lesion and AA with excess inflammatory cells and vascular permeability vs. ApoE-/- mice. By contrast, Atox1-/-/ApoE-/- mice exhibit significant reduction of atherosclerotic lesion, suggesting that ATP7A is atheroprotective while Atox1 is proatherogenic. Of note, both ATP7Amut and Atox1-/- mice show the similar extent of reduced Cu enzyme lysyl oxidase activity mediated through Cu chaperone Atox1, which does not explain enhancing atherosclerosis or AA in ATP7Amut/ApoE-/- mice. Based on additional new preliminary data with ATP7A-depleted ECs, we will test the novel hypothesis that Cu exporter ATP7A downregulation by inflammation increases intracellular Cu that stimulates "nuclear Atox1"- mediated EC barrier dysfunction and ROS-dependent inflammatory responses. This in turn promotes excess inflammatory cell recruitment, which accelerates atherosclerosis and AA. Aim 1 will define the protective role of ATP7A against inflammation-induced endothelial barrier dysfunction and ROS-dependent inflammatory adhesion molecule expression, which contribute to leukocyte transendothelial migration (TEM) in ECs in a Cu- and Atox1-dependent manner. Aim 2 will determine how inflammation-induced accumulated intracellular Cu promotes endothelial barrier dysfunction and ROS-dependent inflammatory responses by focusing on Cu-dependent transcription factor function of Atox1 to upregulate miR125b that represses VE- cadherin as well as p47phox of NADPH oxidase that increases ROS-NFkB. Aim 3 will define the protective role of ATP7A against atherosclerosis and AA development via regulating vascular permeability and inflammation in a Cu- and Atox1-dependent manner in vivo. We will use ATP7Amut, ATP7A transgenic, Atox1-/- mice; or inducible EC-specific ATP7A conditional deficient mice crossed with ApoE-/- mice with high fat diet treated with Cu chelators or LNA-anti-miR125b. Moreover, Cu imaging analysis (64Cu labeling, inductively coupled plasma mass spectrometry (ICP-MS) and synchrotron X-ray fluorescence microscopy (XFM)), live cell imaging, in vivo intravital microscopy, non-invasive Bioluminescence imaging using NFkB transgenic reporter mice will be used. Our study will provide novel insight into Cu transporter ATP7A or nuclear Atox1 as potential therapeutic targets for treatment of vascular inflammatory diseases such as atherosclerosis.

描述（由申请人提供）：动脉粥样硬化受炎症，内皮细胞（EC）屏障功能障碍和氧化应激调节，是主动脉动脉瘤（AA）（AA）的主要原因，以及退伍军人人群中的死亡率。铜（CU）是一种必需的营养素，参与正常功能，而超过CU则导致炎症性疾病，包括具有未知机制的动脉粥样硬化。由于超过Cu是有毒的，因此细胞内Cu的生物利用度通过转运ATPase（ATP7A）的Cu严格控制，该Cu从胞质Cu Chaperone Atox1中获得Cu，然后将CU传递给包括Ecsod的Cu秘书，包括Ecsod，或将Cu extort cu出口到外细胞外空间。我们的实验室报告说，血管ATP7A-ECSOD轴可防止高血压和Type1糖尿病中的内皮功能障碍，并且当ATOX1定位在核时，ATOX1起着CU依赖性转录因子的作用。但是，ATP7A的作用及其与核ATOX1在动脉粥样硬化中的关系完全未知。初步数据表明，与APOE - / - 小鼠杂交的CU输出功能降低的ATP7A突变体（ATP7AMUT）相对于动脉粥样硬化病变加速了，并且具有过量的炎性细胞和血管通透性与APOE - / - 小鼠的AA。相比之下，ATOX1 - / - /APOE-/ - 小鼠表现出明显的动脉粥样硬化病变降低，这表明ATP7A是动脉保护性的，而ATOX1则具有促进性。值得注意的是，ATP7AMUT和ATOX1 - / - 小鼠都表明，通过Cu链酮ATOX1介导的Cu酶氧化物活性降低的程度相似，这不能解释在ATP7AMUT/APOE-/ - 小鼠中增强动脉粥样硬化或AA的AA。基于用ATP7A耗尽的EC进行的其他新初步数据，我们将测试以炎症的CU出口量ATP7A下调的新假设，这会增加细胞内CU，从而刺激“核ATOX1” - 介导的EC障碍性功能障碍和ROS依赖性炎症反应。反过来，这促进了多余的炎性细胞募集，从而加速了动脉粥样硬化和AA。 Aim 1 will define the protected role of ATP7A against inflammation-induced endothelial barrier dysfunction and ROS-dependent inflammatory adhesive molecule expression, Aim 2 will determine how inflammation-induced accumulated intracellular Cu promotes endothelial barrier dysfunction and ROS-dependent inflammatory responses by focusing on Cu-dependent transcription factor function of Atox1 to update miR125b that reflects VE-钙粘着蛋白以及增加ROS-NFKB的NADPH氧化物的P47Phox。 AIM 3将通过体内调节血管通透性和炎症来定义ATP7A对动脉粥样硬化和AA发育的保护作用。我们将使用ATP7AMUT，ATP7A转基因，ATOX1 - / - 小鼠；或可诱导的EC特异性ATP7A有条件的缺乏小鼠与ApoE - / - 小鼠，用Cu螯合剂或LNA-ANTI-MIR125B处理高脂肪饮食。此外，CU成像分析（64CU标记，电感耦合等离子体质谱（ICP-MS）和同步加速器X射线荧光显微镜（XFM）），实时细胞成像，体内室内显微镜检查，使用NFKB Transporter Miles Mime c。我们的研究将提供有关CU转运蛋白ATP7A或核ATOX1作为治疗血管炎性疾病（如动脉粥样硬化）的潜在治疗靶标的新颖的见解。